Multi-plasmid method for preparing large libraries of polyketides and non-ribosomal peptides

ABSTRACT

A multiple-plasmid system for heterologous expression of polyketides facilitates combinatorial biosynthesis. The method can be extended to any modular polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS or NRPS products by tailoring enzymes.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and is, acontinuation-in-part of U.S. Ser. No. 09/422,073, filed Oct. 21, 1999,which is a continuation of U.S. Ser. No. 08/989,332, filed Dec. 11,1997, now U.S. Pat. No. 6,033,883, which claims priority to U.S. Ser.No. 60/033,193, filed Dec. 18, 1996, lapsed; and this application claimspriority to U.S. provisional application Serial No. 60/129,731, filedApr. 16, 1999. Each of the above patent applications is incorporatedherein by reference.

REFERENCE TO GOVERNMENT FUNDING

[0002] This invention was supported in part by SBIR grant1R43-GM56575-01. The U.S. government has certain rights in thisinvention.

FIELD OF THE INVENTION

[0003] The present invention provides recombinant DNA compounds and hostcells containing novel polyketide synthase (PKS) genes and novelpolyketides. The invention relates to the fields of chemistry, medicinalchemistry, human and veterinary medicine, molecular biology,pharmacology, agriculture, and animal husbandry.

BACKGROUND OF THE INVENTION

[0004] Polyketides are structurally diverse natural products thatinclude important therapeutic agents used as antibacterials(erythromycin), immunosuppressants (FK506), cholesterol lowering agents(lovastatin), and others (see Katz et al., 1993, Polyketide synthesis:prospects for hybrid antibiotics, Annu. Rev. Microbiol. 47: 875-912,incorporated herein by reference). Currently, there are about 7,000identified polyketides, but this represents only a small fraction ofwhat nature is capable of producing.

[0005] DNA sequencing of genes encoding several of the enzymes thatproduce type 1 modular polyketide synthases (PKSs) has revealed theremarkably logical organization of these multifunctional enzymes (seeCortes et al., 1990, An unusually large multifunctional polypeptide inthe erythromycin-producing polyketide synthase of Saccharopolysporaerythraea, Nature 348: 176-178; Donadio et al., 1991, Modularorganization of genes required for complex polyketide biosynthesis,Science 252: 675-679; Schwecke et al., 1995, The biosynthetic genecluster for the polyketide immunosuppressant rapamycin, Proc. Natl.Acad. Sci. USA 92: 7839-7843; and August et al., 1998, Biosynthesis ofthe ansamycin antibiotic rifamycin: deductions from the molecularanalysis of the rif biosynthetic gene cluster of Amycolatopsismediterranei S699, Chem Biol 5: 69-79, each of which is incorporatedherein by reference). The application of innovative combinatorialtechniques to this genetic organization has prompted the generation ofnovel natural products, by adding, deleting, or exchanging domains orentire modules. See U.S. Pat. Nos. 5,672,491; 5,712,146; 5,830,750;5,843,718; 5,962,290; and 6,022,731, each of which is incorporatedherein by reference. It would be advantageous to have a practicalcombinatorial biosynthesis technology that could achieve and perhapsexceed the diversity of modular polyketide structures thus far revealedin nature.

[0006] The known modular PKSs have a linear organization of modules,each of which contains the activities needed for one cycle of polyketidechain elongation, as illustrated for 6-deoxyerythronolide B synthase(DEBS) in FIG. 1A. The minimal module contains a ketosynthase (KS), anacyltransferase (AT), and an acyl carrier protein (ACP) that togethercatalyze a 2-carbon extension of the chain. The specificity of the ATfor either malonyl or an alpha-alkyl malonyl CoA determines which2-carbon extender is used, and thus the nature of the alkyl substituentat the alpha-carbon of the growing polyketide chain. After each 2-carbonunit condensation, the oxidation state of the beta-carbon is eitherretained as a ketone, or modified to a hydroxyl, methenyl, or methylenegroup by the presence a ketoreductase (KR), a KR+ a dehydratase (DH), ora KR+DH+ an enoyl reductase (ER), respectively. In effect, the ATspecificity and the composition of catalytic domains within a moduleserve as a “code” for the structure of each 2-carbon unit. The order ofthe modules in a PKS specifies the sequence of the distinct 2-carbonunits, and the number of modules determines the size of the polyketidechain.

[0007] The remarkable structural diversity of polyketides (see O'Hagan,The Polyketide Metabolites; Ellis Horwood, Chichester, 1991,incorporated herein by reference) is governed by the combinatorialpossibilities of arranging modules containing the various catalyticdomains, the sequence and number of modules, and the post-PKS “tailoringenzymes” that accompany the PKS genes. The direct correspondence betweenthe catalytic domains of modules in a PKS and the structure of theresulting biosynthetic product allows rational modification ofpolyketide structure by genetic engineering.

[0008] Over the past several years, examples of modifying each of theelements that code for polyketide structure has been accomplished (seeKao et al., 1996, Evidence for two catalytically independent clusters ofactive sites in a functional modular polyketide synthase, Biochemistry35: 12363-12368; Liu et al., 1997, Biosynthesis of2-nor-6-deoxyerythronolide B by rationally designed domain substitution,J. Am. Chem. Soc. 119: 10553-10554; McDaniel et al., 1997,Gain-of-function mutagenesis of a modular polyketide synthase, J. Am.Chem. Soc. 119: 4309-4310; Marsden et al., 1998, Engineering broaderspecificity into an antibiotic-producing polyketide synthase, Science279: 199-202; and Jacobsen et al., 1997, Precursor-directed biosynthesisof erythromycin analogs by an engineered polyketide synthase, Science277: 367-369, each of which is incorporated herein by reference).

[0009] Recently, a combinatorial library of over 50 novel polyketideswas prepared by systematic modification of DEBS, the PKS that producesthe macrolide aglycone precursor of erythromycin (see U.S. patentapplication Ser. No. 09/429,349, filed Oct. 28, 1999; PCT patentapplication US99/24483, filed Oct. 20, 1999; and McDaniel et al., 1999,Multiple genetic modification of the erythromycin gene cluster toproduce a library of novel “unnatural” natural products, “Proc. Natl.Acad. Sci. ” USA 96: 1846-1851, each of which is incorporated herein byreference). With a single plasmid containing the eryAI, -AII and -AIIIgenes encoding the three DEBS subunits, ATs and beta-carbon processingdomains were substituted by counterparts from the rapamycin PKS (seeSchwecke et al., 1995, supra) that encode alternative substratespecificities and beta-carbon processing activities. The approach usedwas to develop single “mutations”, then sequentially combine the singlemutations to produce multiple changes in the PKS. It was observed thatwhen two or more single PKS mutants were functional, there was a highlikelihood that combinations would also produce the expected polyketide.Although this strategy provided high assurance that the multiple mutantswould be productive, the production of each polyketide required aseparate engineering. Thus, if X mutants of eryAI, Y mutants of eryAII,and Z mutants at eryAIII were prepared, X+Y+Z separate experiments wererequired to produce that same number of polyketides. Clearly, thepreparation of very large libraries by this approach is laborious.

[0010] Another strategy for preparing large numbers of polyketides is byrandom digestion-religation leading to “mutagenesis” of the domains ormodules of a mixture of PKS genes, including the refinements embodied inthe DNA shuffling method (see Patten et al., 1997, Applications of DNAshuffling to pharmaceuticals and vaccines, Curr. Op. Biotechnol. 8:724-733, incorporated herein by reference). The expected low probabilityof assembling an active PKS by such an approach, however, would demandan extraordinary analytical effort (in the absence of a biologicalselection) to detect clones that produced polyketides within the muchlarger number of clones that are non-producers.

[0011] There remains a need for practical approaches to create largelibraries of polyketides, non-ribosomal peptides, and mixedpolyketides/non-ribosomal peptides.

SUMMARY OF THE INVENTION

[0012] The present invention provides a method for expressing apolyketide or non-ribosomal peptide in a host cell employing amultiplicity of recombinant vectors, which may be integrative or freelyreplicating. Each of the multiplicity of vectors encodes a portion ofthe polyketide synthase or non-ribosomal peptide synthase that producesthe polyketide or non-ribosomal peptide. In one embodiment, at least oneof the multiplicity of vectors encodes one or more proteins that furthermodify the polyketide or non-ribosomal peptide produced.

[0013] In a preferred embodiment, the vectors replicate in and/orintegrate into the chromosome of a Streptomyces host cell. Preferredintegrating vectors include vectors derived from pSET152 and pSAM2.Preferred replicating vectors include those containing a repliconderived from SCP2* or pJV1.

[0014] In another embodiment, the present invention provides novelpolyketides. Such novel polyketides include those shown in FIG. 3 ascompound nos. 29-43 and 45-59. Other novel polyketides of the inventioninclude the polyketides obtainable by hydroxylation and/or glycosylationof compounds 2943 and 45-59. Preferred compounds of the inventioninclude those 14-membered macrolactones with a C-6 and/or C-12 hydroxyland/or a C-3 and/or C-5 glycosyl, including but not limited to thosewith a desosaminyl residue at C-5 and a cladinosyl residue at C-3.

[0015] These and other embodiments, modes, and aspects of the inventionare described in more detail in the following description, the examples,and claims set forth below.

DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows wild-type and mutant forms of the eryA genes and DEBSproteins. Part A depicts the eryAI-eryAIII genes and proteins as broadarrows oriented in the direction of transcription with the domains inmodules 1 to 6 of DEBS1-DEBS3 indicated by the symbols defined herein.The first substrate, propionyl-CoA, is attached to the loading domainACP and (2S)-2-methylmalonyl-CoA to the module 2 ACP. Then, adecarboxylative condensation between the propionate and methylmalonatetakes place followed by reduction of the incipient beta-ketone to formthe intermediate shown attached to the ACP of module 2. Thisintermediate is transferred to the ACP of module 3, and the sequence ofreactions is repeated at each of the other modules with or withoutketone reduction, dehydration, or double bond reduction to form thelinear 21-carbon polyketide attached to the ACP of module 6. The linearpolyketide then is cyclized and released as 6-deoxyerythronolide B(6dEB). Part B depicts replacement of the one or more of the domains inDEBS1, DEBS2, or DEBS3 with one of the three rap (rapamycin) PKS domainsor cassettes, or deletion of the KR. This results in the correspondingfunctional group changes shown at one or more of the positions of 6dEB.

[0017]FIG. 2 shows an illustrative three-plasmid expression system foreryA genes. In each vector, the eryA gene is expressed under the controlof the upstream actI promoter and actII-ORF4 gene as previouslydescribed (see Ziermann et al., 2000, A two-vector system for theproduction of recombinant polyketides in Streptomyces, J. Ind.Microbiol. & Biotech. 24: 46-50; and Kao et al., 1994, Engineeredbiosynthesis of a complete macrolactone in a heterologous host, Science265: 509-512, each of which is incorporated herein by reference). Tofacilitate construction of the various eryA mutations, a SpeI site(ACTAGT) was introduced at nt 10366-10371 of the eryAI ORF by makingD3455T and A3456S mutations (see Kao et al., 1995, Manipulation ofmacrolide ring size by directed mutagenesis of a modular polyketidesynthase, J. Am. Chem. Soc. 117: 9105-9106, incorporated herein byreference) in pKOSO25-179. This change enables insertion of the mutatedgene segment between the PacI site and the Spel site of pKOS025-179.After the replacement of the 6-kb fragment between the AscI sites ineryAII (nt 1213 and nt 7290) with a 6-kb AscI fragment containing aspecific AT substitution in an intermediate plasmid, the resultingPacI-XbaI fragment containing the mutant eryAII gene was inserted intopKOS025-143. AII eryAIII mutants were constructed by replacing thesegment in pKOS010-153 between the unique BglII site at nt 251 and theEcoRI site (nt 9290) that overlaps the stop codon.

[0018]FIG. 3 shows structures of macrolactones produced by Streptomyceslividans strains containing assorted combinations of three plasmids(FIG. 2 and Table 1). The positions in 6dEB (1) that are alteredcorrespond to the genetic characteristics of modules 2, 3, 5 and 6 ofDEBS, as illustrated in FIG. 1A and FIG. 2, and listed in Table 1.Structures 1-40 and 49-56 are 14-membered lactones, and structures 44 to48 are 12-membered lactones. Compounds 49-59 with the C13 propyl groupwere produced by mutational biosynthesis with the eryAI KS1° nullallele.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides methods and reagents for usingmultiple recombinant DNA vectors to produce polyketides in recombinanthost cells. In an illustrative embodiment, a three-plasmid system forheterologous expression of 6-deoxyerythronolide B synthase (DEBS) isprovided to facilitate combinatorial biosynthesis of polyketides made bytype I modular polyketide synthases (PKSs). The eryA PKS genes encodingthe three DEBS subunits were individually cloned into three compatibleStreptomyces vectors carrying mutually selectable antibiotic resistancemarkers. A strain of Streptomyces lividans transformed with all threeplasmids produced 6-deoxyerythronolide B at a level similar to that of astrain transformed with a single plasmid containing all three genes.

[0020] The utility of this system in combinatorial biosynthesis wasdemonstrated through production of a library of modified polyketidemacrolactones, using versions of each plasmid constructed to containdefined mutations. Combinations of these vector sets were introducedinto Streptomyces lividans, resulting in strains producing a wide rangeof 6-deoxyerythronolide B analogs. This method can be applied to anymodular PKS or non-ribosomal peptide synthase (NRPS) and has thepotential to produce thousands of novel natural products, including onesderived from further modification of the PKS or NRPS products bytailoring enzymes.

[0021] Thus, in one aspect, the present invention provides a method forusing multiple (two, three, four, or more) recombinant DNA vectors, eachencoding a portion of a PKS, NRPS, or tailoring enzyme, to produce apolyketide or non-ribosomal peptide or a mixed polyketide/non-ribosomalpeptide in a host cell. In one embodiment, the vectors in combinationencode a naturally occurring PKS or NRPS and the corresponding naturalproduct is produced. In another embodiment, the vectors in combinationencode naturally occurring proteins from two or more different naturallyoccurring PKS or NRPS. In another embodiment, at least one of thevectors encodes a protein not found in nature, having been altered byrecombinant DNA methodology to change its structure and function.

[0022] In one embodiment, the present invention provides a method forcreating a polyketide library that enables the production of largelibraries of polyketides while retaining the high probability ofobtaining productive clones by combining PKS mutations known to beproductive. The principle involves cloning mutants of individual openreading frames (ORFs) of a PKS on separate compatible plasmids, thencoexpressing the separate ORFs in a suitable host to produce the PKS.Using this multiple plasmid approach, with X mutants of ORF 1, Y mutantsof ORF 2, and Z mutants of ORF 3, for instance, a combinatorial libraryof X×Y×Z mutants can be achieved expeditiously.

[0023] DEBS was chosen to illustrate this multiple plasmid approach. TheDEBS PKS consists of three >280 kD protein subunits, each containing twomodules, that are assembled into the complete PKS complex (see Donadioet al., 1991, supra). With the two possible AT domains and the fourpossible beta-keto modifications, i.e. eight at each module, therefore8×8=64 permutations for the two modules are possible in each DEBSsubunit. Constructing the mutations in each DEBS ORF separately wouldrequire that 64 manipulations be carried out on each gene, or a total of192 such manipulations. However, by co-transforming a host strain withthree plasmids, each bearing the 64 permutations of a different DEBSsubunit, one could generate the mutant PKSs necessary to achieve, intheory, a library of 262,144 polyketides (64³), as 6-deoxyerythronolideB (6dEB) analogs (FIG. 1A). In contrast, the same number of mutagenesisexperiments performed in a single plasmid system would theoreticallyyield only 192 polyketides.

[0024] The successful implementation of this multiple plasmid strategyrequires that the DEBS subunits translated from three different mRNAsfaithfully interact to give the active PKS. Some indication that thiswould be the case was provided by in vitro experiments that showed thatreconstitution of the isolated DEBS1-DEBS2 complex with DEBS3 forms afunctional PKS (see Pieper et al., 1995, Cell-free synthesis ofpolyketides by recombinant erythromycin polyketide synthases, Nature378: 263-266, incorporated herein by reference). Further, it wasrecently demonstrated that coexpression of the three subunits of DEBSfrom two plasmids produced active PKS in vivo (see Ziermann et al.,2000, supra). The present invention can be applied to any PKS or NRPS,including but not limited to the PKS enzymes, including the KS1 nullmutation containing versions, that synthesize oleandolide andmegalomicin (see U.S. patent application Serial No. 60/158,305, filedOct. 08, 1999 and Ser. No. 09/428,517, filed Oct. 28, 1999, and PCTapplication No. US99/24478, filed Oct. 22, 1999, each of which isincorporated herein by reference).

[0025] The method in one embodiment requires at least three vectors thatcan be separately introduced into a Streptomyces or other suitable hoststrain and concomitantly express functional PKS subunits. Two suchvectors are preferred for such purposes: the autonomously replicatingSCP2*-based plasmid pRM1 (see Kao et al., 1994, Engineered biosynthesisof a complete macrolactone in a heterologous host, Science 265: 509-512,and U.S. Pat. No. 6,022,731, each of which is incorporated herein byreference) and the integrating bacteriophage phiC31-based plasmidpSET152 (see Bierman et al., 1992, Plasmid cloning vectors for theconjugal transfer of DNA from Escherichia coli to Streptomyces spp.,Gene 116:43-49, incorporated herein by reference). Several additionalplasmids have been tested as described herein and can be used inaccordance with the present invention.

[0026] Each of these additional plasmids was tested using constructsthat contained identical configurations of an eryA gene downstream ofthe Streptomyces coelicolor acti promoter and actII-ORF4 transcriptionalactivator as described (see Ziermann et al., 2000, supra). Plasmid pB45,a high copy replicating plasmid possessing the pJv1 origin (seeServin-Gonzalez et al., 1995, Sequence and functional analysis of theStreptomyces phaechromogenes plasmid pJV1 reveals a modular organizationof Streptomyces plasmids that replicate by rolling circle, Microbiology141: 2499-2510, incorporated herein by reference) carrying eryAII wasintroduced into S. lividans harboring eryAI on pRM1 and eryAIII in thepSET152 integration site; less than 0.1 mg/L of 6dEB was produced inthis system compared with 50 mg/L for the single plasmid system (see Kaoet al., 1994, supra).

[0027] The multiple vectors used in the present method can include twoor more different vectors that share the same origin of replication. Forexample, two SCP2*-type plasmids carrying different antibiotic markerscan coexist and express PKS subunits in a Streptomyces sp., as has beenreported using high-copy number plasmids (see Rajgarhia et al., 1997,Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesisgenes required for aklanonic acid biosynthesis, J. Bacteriol. 179:2690-2696, incorporated herein by reference). Co-transformation of S.lividans with eryAIII/pSET-apm, eryAI/pRM1-tsr and eryAII/pRM1-hyg (FIG.2) yielded a strain that also produced 50 mg/L of 6dEB. Further, afterover 24 generations under double antibiotic selection, both replicatingplasmids could be rescued by standard procedures with unchangedrestriction maps (see Hopwood et al., 1985, Genetic Manipulation ofStreptomyces. A laboratory manual. John Innes Foundation, Norwich,incorporated herein by reference).

[0028] The multiple vectors of the present invention can include two ormore different integrating vectors as well. For example, the eryAII genewas cloned into a pSAM2 site-specific integrating plasmid (see Smokvinaet al., 1990, Construction of a series of pSAM2-based integrativevectors for use in Actinomycetes, Gene 94: 53-59, incorporated herein byreference). Sequential transformation of Streptomyces lividans witheryAIII/pSET-apm, eryAII/pSAM-hyg and eryAI/pRM1-tsr provided a strainthat produced 40-50 mg/L of 6dEB. A potential advantage of this systemover the two-replicating vector system is that it requires one fewerantibiotic, and avoids potential problems in maintaining two plasmidscontaining the same ori in a Streptomyces host (see Baltz, 1997,Molecular genetic approaches to yield improvement in Actinomycetes,Biotechnology of Antibiotics, 2nd. Edition: pp. 49-62, incorporatedherein by reference). However, as shown herein, the two SCP2*-basedplasmids and the pSET-derived vector can also be used.

[0029] Thus, the invention can be practiced with a wide variety ofexpression vectors for use in Streptomyces. The replicating expressionvectors of the present invention include, for example and withoutlimitation, those that comprise an origin of replication from a low copynumber vector, such as SCP2* (see Hopwood et al., Genetic Manipulationof Streptomyces: A Laboratory manual (The John Innes Foundation,Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieserand Melton, 1988, Gene 65: 83-91, each of which is incorporated hereinby reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62,incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol.Gen. Genet. 219: 341-348, and Bierman et al., 1992, Gene 116:43-49, eachof which is incorporated herein by reference), or a high copy numbervector, such as pIJ101 and pJV1 (see Katz et al., 1983, J. Gen.Microbiol. 129: 2703-2714; Vara et al., 1989, J. Bacteriol. 171:5782-5781; and Servin-Gonzalez, 1993, Plasmid 30: 131-140, each of whichis incorporated herein by reference). High copy number vectors are,however, generally not preferred for expression of large genes ormultiple genes. For non-replicating and integrating vectors andgenerally for any vector, it is useful to include at least an E. coliorigin of replication, such as from pUC, p1P, p1I, and pBR. For phagebased vectors, the phage phiC31 and its derivative KC515 can be employed(see Hopwood et al., supra). Also, plasmid pSET152, plasmid pSAM,plasmids pSE101 and pSE211, all of which integrate site-specifically inthe chromosomal DNA of S. lividans, can be employed for purposes of thepresent invention.

[0030] Moreover, a wide variety of selectable markers can be employed inthe Streptomyces recombinant expression vectors of the invention. Theseinclude antibiotic resistance conferring genes selected from the groupconsisting of the ermE (confers resistance to erythromycin andlincomycin), tsr (confers resistance to thiostrepton), aadA (confersresistance to spectinomycin and streptomycin), aacC4 (confers resistanceto apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin),hyg (confers resistance to hygromycin), and vph (confers resistance toviomycin) resistance conferring genes. Alternatively, severalpolyketides are naturally colored, and this characteristic can provide abuilt-in marker for identifying cells.

[0031] An illustrative library of the present invention was constructed.The library was composed of vectors encoding three single mutations ineryAI (module 2), one in eryAII (module 3), and seven in eryAIII(modules 5 or 6) as well as wild-type ORFs, a KS1 null mutant, and amodule 6 deletion, as shown in Table 1, below. To facilitate cloning,vectors were prepared that contained restriction sites that allowedtransfer of DNA cassettes from previously prepared mutant eryA genes(see McDaniel et al., 1999, supra, and FIG. 2). Fourteen of theseexpression vectors, comprising the three wild-type and eleven mutantORFs were constructed by cassette transfers from plasmids previouslyprepared in the single plasmid system. TABLE 1 Genotype of the plasmidscontaining DEBS genes^(a) Vector: pKOS021 pKOS025 pKOS010 Gene:eryAI(DEBS1) eryAII(DEBS2) eryAIII(DEBS3) Module: 1 3 5 6 1. wild-type2. AT->^(b)rapAT2 3. KR->rapDH/KR4 4. KR->rapDH/ER/KR1 5. KS1°^(c) 6.wild-type 7. AT3->rapAT2  8. wild-type  9. AT->rapAT2 10. KR->AT/ACPlinker 11. KR->rapDH/KR4 12. KR->rapDH/ER/KR1 13. module 5 + TE^(d) 14.AT->rapAT2 15. KR->AT/ACP linker 16. KR->rapDH/KR4

[0032] In eryAI, module 2 was modified by replacing the AT by rapAT2(the AT domain of module 2 of the rapamycin PKS), and the KR byrapDH/KR4 (the DH and KR domains of module 4 of the rapamycin PKS) orrapDH/ER/KR1 (the DH, KR, and ER domains of module 1 of the rapamycinPKS). In eryAII, the AT of module 3 was replaced by rapAT2; in eryAIII,module 5 was modified by replacing the AT by rapAT2, and the KR byrapDH/KR4 or rapDH/ER/KR1 or the AT/ACP linker that eliminates the erymodule 5 KR activity. Also, in module 6 the AT was replaced with rapAT2,and the KR by rapDH/KR4 or the AT/ACP linker. The consequence ofintroducing each of these rapamycin PKS gene cassettes into DEBS isshown in FIG. 1B; at a given alpha-position in the growing polyketidecarbon chain, a methyl group can be removed, or a beta-hydroxyl can bechanged to a ketone or removed by dehydration to produce a double bond,or the double bond resulting from dehydration can then be reduced to amethylene group.

[0033] The approach employed was to first introduce the eight eryAIIIvariants individually into the pSET integration site of S. lividans,then to cotransform the resulting strains individually with each of foureryAI variants on SCP2* -tsr vectors and two eryAII variants onSCP2*-hyg vectors. Thus, from the 14 vectors prepared, 64 tripletransformants were obtained. Recombinant cells were grown underappropriate antibiotic selection, and extracts analyzed for polyketideproduction by LC/MS. Of the 64 triple transformants, 46 (72%) produceddetectable levels of one or more polyketides under the test conditionsemployed (FIG. 3), and 43 different polyketides were produced. Theseincluded 6dEB (1), and products arising from 11 single (2-12), 26 double(13-38) and five (39-43) triple mutants of DEBS. Twenty-eight (1-28) ofthe 43 polyketides produced have been previously prepared using thesingle-plasmid system. Fifteen (29-43) were novel polyketides readilyidentifiable by mass spectra and correspondence to the products expectedbased on the cassettes used in the mutagenesis.

[0034] In one aspect, the present invention provides these novelpolyketides. These novel polyketides can be further modified bytailoring enzymes, including the tailoring enzymes of Saccharopolysporaerythraea. There are a wide variety of diverse organisms that can modifymacrolide aglycones to provide compounds with, or that can be readilymodified to have, useful activities. For example, Saccharopolysporaerythraea can convert 6-dEB or derivatives thereof to a variety ofuseful compounds. The erythronolide 6-dEB is converted by the eryf geneproduct to erythronolide B, which is, in turn, glycosylated by the eryBgene product to obtain 3-O-mycarosylerythronolide B, which containsL-mycarose at C-3. The enzyme eryc gene product then converts thiscompound to erythromycin D by glycosylation with D-desosamine at C-5.Erythromycin D, therefore, differs from 6-dEB through glycosylation andby the addition of a hydroxyl group at C-6. Erythromycin D can beconverted to erythromycin B in a reaction catalyzed by the eryG geneproduct by methylating the L-mycarose residue at C-3. Erythromycin D isconverted to erythromycin C by the addition of a hydroxyl group at C-12in a reaction catalyzed by the eryK gene product. Erythromycin A isobtained from erythromycin C by methylation of the mycarose residue in areaction catalyzed by the eryG gene product. The aglycone compoundsprovided by the present invention, such as, for example, the compoundsproduced in Streptomyces lividans, can be provided to cultures of S.erythraea and converted to the corresponding derivatives oferythromycins A, B, C, and D. To ensure that only the desired compoundis produced, one can use an S. erythraea eryA mutant that is unable toproduce 6-dEB but can still carry out the desired conversions (Weber etal., 1985, J. Bacteriol. 164(1): 425-433). Also, one can employ othermutant strains, such as eryB, eryC, eryG, and/or eryK mutants, or mutantstrains having mutations in multiple genes, to accumulate a preferredcompound. The conversion can also be carried out in large fermentors forcommercial production.

[0035] Moreover, there are other useful organisms that can be employedto hydroxylate and/or glycosylate the compounds of the invention. Asdescribed above, the organisms can be mutants unable to produce thepolyketide normally produced in that organism, the fermentation can becarried out on plates or in large fermentors, and the compounds producedcan be chemically altered after fermentation. Thus, Streptomycesvenezuelae, which produces picromycin, contains enzymes that cantransfer a desosaminyl group to the C-5 hydroxyl and a hydroxyl group tothe C-12 position. In addition, S. venezuelae contains a glucosylationactivity that glucosylates the 2′-hydroxyl group of the desosaminesugar. This latter modification reduces antibiotic activity, but theglucosyl residue is removed by enzymatic action prior to release of thepolyketide from the cell. Another organism, S. narbonensis, contains thesame modification enzymes as S. venezuelae, except the C-12 hydroxylase.Thus, the present invention provides the compounds produced byhydroxylation and glycosylation of the macrolide aglycones of theinvention by action of the enzymes endogenous to S. narbonensis and S.venezuelae.

[0036] Other organisms suitable for making compounds of the inventioninclude Micromonospora megalomicea, which produces megalomicin A.Megalomicin A contains the complete erythromycin C structure, and itsbiosynthesis also involves the additional formation of megosamine(L-rhodosamine) and its attachment to the C-6 hydroxyl, followed byacylation of the C-3′″ and(or) C-4′″ hydroxyls as the terminal steps.Other organisms useful in converting the aglycones of the presentinvention to modified compounds include Streptomyces antibioticus, S.fradiae, and S. thermotolerans. S. antibioticus produces oleandomycinand contains enzymes that hydroxylate the C-6 and C-12 positions,glycosylate the C-3 hydroxyl with oleandrose and the C-5 hydroxyl withdesosamine, and form an epoxide at C-8-C-8a. S. fradiae contains enzymesthat glycosylate the C-5 hydroxyl with mycaminose and then the4′-hydroxyl of mycaminose with mycarose, forming a disaccharide. S.thermotolerans contains the same activities as S. fradiae, as well asacylation activities. Thus, the present invention provides the compoundsproduced by hydroxylation and glycosylation of the macrolide aglyconesof the invention by action of the enzymes endogenous to S. antibioticus,S. fradiae, and S. thermotolerans. Moreover, these and other tailoringenzymes can be cloned and incorporated into one or more of the multiplevectors used in accordance with the methods of the present invention tocreate libraries of modified polyketide compounds.

[0037] The resulting compounds can be further modified by syntheticchemistry, i.e., to yield the corresponding ketolides, useful asantibiotics (see, e.g., U.S. patent application Serial No. 60/172,159,filed Dec. 17, 1999; No. 60/140,175, filed Jun. 18, 1999; No.60/172,154, filed Dec. 17, 1999; and No. 60/129,729, filed Apr. 16,1999, each of which is incorporated herein by reference), or to yieldthe corresponding motilides (see U.S. provisional patent applicationSerial No. 60/183,338, filed Feb. 18, 2000, attorney docket no.30062-30053.00, inventors G. Ashley et al., incorporated herein byreference).

[0038] In 43 of the 46 transformants that produced polyketides in theillustrative library described herein, the isolated polyketides hadstructures expected of the mutations). However, as observed incorresponding single mutations of the eryA gene in the single-plasmidsystem, additional products were observed with certain mutations. Inmost cases, the rapAT2 domain in module 3 recognized and processed bothmalonyl- and methylmalonyl-CoA and gave the expected 8-nor analogs pluslesser amounts of the 8-methyl analogs. In a few cases, only the8-methyl analogs were formed. The relaxed-specificity of rapAT2 appearsto be module-dependent, because only the expected products were observedwith the same rapAT2 sequence at modules 2 or 5 (see Ruan et al., 1997,Acyltransferase domain substitutions in erythromycin polyketide synthaseyield novel erythromycin derivatives, J. Bacteriol. 179: 6416-25,incorporated herein by reference).

[0039] Likewise, when the KR of module 5 was replaced by either therapDH/KR4 or rapDH/ER/KR1 domains to give the expected 4,5-anhydro and5-deoxy analogs, respectively, 5-keto analogs were formed in addition tothe expected products. This possibly results from transfer of thebeta-ketothioester intermediate from KR5 to ACP5 at a rate competitivewith its reduction by the heterologous rapKR4 domain. Because aberrantproducts were not observed with rapDH/KR4 at modules 2 or 6, thenon-specificity appears to be module-dependent.

[0040] Finally, when the KR of module 2 was replaced with therapDH/ER/KR1 domain to provide the 11-deoxy analogs, the 10,11-dehydroanalogs were often observed as minor but significant products. Here, theintermediate is processed by the heterologous rapKR and DH domains inmodule 2, but transfer of the 10,11-dehydro intermediate to KS3 must becompetitive with the ER-catalyzed reduction. Interestingly, withrapDH/ER/KR1 in module 2 and rapAT2 in module 3, the 10,11-dehydroby-products were not detected. Either the levels produced were too lowfor detection using the methods employed, or the aberrant dehydrointermediate of module 2 was not processed by module 3 containing therapAT2 substitution.

[0041] Of the 18 transformants that did not produce polyketides atlevels detectable in these experiments, two were double mutants and 16were triple mutants; only one of these mutants was previously preparedin the single-plasmid system where it also failed to produce adetectable polyketide. As previously reported with the single-plasmidsystem, an increased number of mutations resulted in a decrease in yieldto a level undetectable by the analytical method used.

[0042] A major advantage of the multiple plasmid system is that oncemultiple plasmids encoding functional mutants of PKS subunits areavailable, they can be rapidly combined with one or more additionalmutants to expand the library of polyketides. In one example, a singleCys729Ala mutation at the KS1 domain of DEBS1 module 1 (see Jacobsen etal., 1997, supra) was prepared, yielding the KS1 null mutation. Theinactive KS1 prevents propagation of the starter unit and permitsintroduction of exogenous synthetic diketide thiol esters into positions12 and 13 of the 14-membered macrolide product. This system was firstdeveloped using a single vector, plasmid pJRJ2. Plasmid pJRJ2 encodesthe eryAI, eryAII, and eryAIII genes; the eryAI gene contained in theplasmid contains the KS1 null mutation. The KS1 null mutation preventsformation of the 6-deoxyerythronolide B produced by the wild-type geneunless exogenous substrate is provided. Plasmid pJRJ2 and a process forusing the plasmid to prepare novel 13-substituted erythromycins aredescribed in PCT publication Nos. 99/03986 and 97/02358 and in U.S.patent application Ser. No. 08/675,817, filed Jul. 5, 1996; Ser. No.08/896,323, filed Jul. 17, 1997; and Ser. No. 09/311,756, filed May 14,1999, each of which is incorporated herein by reference. The exogenoussubstrates provided can be prepared by the methods and include thecompounds described in PCT patent application No. PCT/ US00/02397(Attorney Docket No. 30062-20032.40) and U.S. patent application Ser.No. 09/492,733 (Attorney Docket No. 30062-20032.00), both filed Jan. 27,2000, by inventors G. Ashley et al., and both of which claim priority toU.S. patent application Serial No. 60/117,384, filed Jan. 27, 1999, eachof which is incorporated herein by reference.

[0043] The plasmid encoding the KS1 null allele of eryAI (Table 1) wasintroduced by co-transformation into Streptomyces lividans with the oneeryAII mutant and seven eryAIII mutants (Table 1) to provide 16transformants. Treatment of each of these with a propyl-diketideN-acetylcysteine thioester, following the work of Jacobsen et al., 1997,and the methodology of the patent applications, supra, provided elevennovel 13-propyl polyketide analogs (49-59, FIG. 3). Thus, the presentinvention provides these novel polyketides as well as the compoundsresulting from their modification by post-PKS tailoring enzymes(oxidases and glycosylases) or by synthetic chemical methods. To preparethese same PKSs by the single-plasmid system would have requiredpreparation of 16 individual mutants rather than the single KS1 nullmutant used in the present method.

[0044] In another example, the variants of eryAI and eryAII were used toprepare a small library of 12-membered macrolactones. It was previouslyshown in the eryA single-plasmid system that omission of module 6, alongwith fusion of module 5 to the thioesterase domain of DEBS3 (FIG. 1A)results in formation of a 12-membered macrolactone. A truncated eryAIIIgene containing module 5-TE (see Kao et al., 1995, Manipulation ofmacrolide ring size by directed mutagenesis of a modular polyketidesynthase, J. Am. Chem. Soc. 117: 9105-9106, incorporated herein byreference, and Table 1) was introduced into Streptomyces lividans, andthe strain transformed with permutations of the four eryAI and twoeryAII variants described above. Of the eight 12-membered lactones thatcould have been produced, five were observed (44-48). Compound 44results from combining the wild-type eryAI and eryAII genes with themodule 5-TE construct, and has previously been prepared in thesingle-plasmid system; compounds 45-48 are novel products that more thandouble the number of known 12-membered macrolides. These novelmacrolactones constitute an important aspect of the present invention.

[0045] Other uses of this multiple plasmid system are as follows. Theexpression and genetic engineering of very large PKS genes, such asthose involved in the biosynthesis of rapamycin (14 modules) orrifamycin (10 modules), is readily achievable by this method. The ORFsfor each of the subunits of these and other PKSs, such as the mixedNRPS-PKS for epothilone (see U.S. patent application Ser. No.09/443,501, filed Nov. 19, 1999, and PCT patent application US99/27438,filed Nov. 19, 1999, each of which is incorporated herein by reference)could be cloned and expressed in the manner used here to greatlysimplify genetic manipulations and the structure/function analysis.Thus, in this application of the present method, a naturally occurringpolyketide can be expressed in a recombinant host cell.

[0046] Similarly, it would be desirable to mix PKS genes from entirelydifferent pathways to facilitate production of heterologous and hybridPKss, as precedented by the work of Li et al. involving hybriderythromycin/picromycin and oleandomycin/picromycinPKS genes (see U.S.patent application Ser. No. 09/320,878, filed May 27, 1999, and Ser. No.09/428,517, filed Oct. 28, 1999; PCT patent publication No. 99/61599;and PCT patent application No. US99/24478, each of which is incorporatedherein by reference). Such work could include extending the carbon chainby the addition of modules.

[0047] The PKS libraries generated could be leveraged and expanded byintroducing genes for tailoring enzymes that oxidize, hydroxylate,methylate, acylate, glycosylate, or otherwise modify the product of thePKS or a modified polyketide, and genes encoding such enzymes could beemployed using an additional vector in the multiple vector system.Various tailoring enzymes from different host organisms could beemployed in the same system. Genes for such tailoring enzymes can beobtained as described in the patent applications and publicationsreferenced in the preceding paragraph and elsewhere herein. See also,U.S. Pat. No. 5,998,194, incorporated herein by reference.

[0048] Finally, the method should be useful with any system consistingof multimodular proteins, such as the large family of non-ribosomalpeptide synthases, which produce many pharmaceutically importantcompounds such as anti-fungal compounds, anti-cancer compounds, andantibiotics. These important compounds are made by multifunctionalsynthetases, consisting of complexes of proteins containing between oneand eleven modules, comparable to the modular PKSs (see Konz et al.,1999, How do peptide synthase generate structural diversity? Chem. &Biol. 6: 39-48, incorporated herein by reference).

[0049] The multi-plasmid technology enables the realization of the fullpotential of modular PKSs and NRPSs, and thus libraries containing acomplete repertoire of polyketides and non-ribosomal peptides. Theachievement of this objective requires only the construction of alimited number of highly expressing, productive single mutants that willassure adequate polyketide production when the mutations are combined.Because all the elements for producing an extraordinarily largepolyketide library are contained within this facile system, there isneither need nor benefit to embark on developing more complicated, lessreliable systems to reach the same objective.

[0050] The following examples are given for the purpose of illustratingthe present invention and shall not be construed as being a limitationon the scope of the invention or claims.

EXAMPLE 1 Construction of Expression Plasmids

[0051] In each vector, the eryA gene is expressed under the control ofthe upstream acti promoter and actII-ORF4 gene as previously described(see U.S. Pat. No. 5,672,491 and Ziermann et al., 2000, supra, both ofwhich are incorporated herein by reference). For testing the high copyrelicating plasmid possessing the pJV1 origin, the pB45 based E. colishuttle vector pKOSO25-32 was constructed by fusing pB45 with litmus 28(New England Biolabs) at BglII and PstI sites. Into pKOS025-32 the ca.14-kb HindIII-XbaI fragment containing the actI promoter and actII-ORF4gene was inserted followed by the eryAII gene to give pKOS025-35. Theconfiguration of the components in the HindIII-XbaI fragment is shown inFIG. 2. The same HindIII-XbaI fragment was also cloned into shuttlevector pOSint1/Hygro (see Raynal et al., 1998, Structure of thechromosomal insertion site for pSAM2: functional analysis in Escherichiacoli, Molecular Microbiology 28: 333-342, incorporated herein byreference) to yield the eryAII/pSAM2-hyg plasmid, pKOS038-67.

EXAMPLE 2 Transfer of Mutation Cassettes into the Three-plasmid System

[0052] The plasmids containing eryAI mutations in module 2 werepKOS025-179 (AT2→rapAT2), pKOS038-1 (KR→rapDH/KR4) and pKOS038-3(KR→rapDH/ER/KR1) and were made as follows. The PacI-SpeI fragmentscontaining the corresponding mutations were transferred into theplasmids described in FIG. 2 from plasmids pKOS008-41, pKOS015-56 andpKOS015-57, respectively (see McDaniel et al., 1999, supra, and U.S.patent application Ser. No. 09/429,349, filed Oct. 28, 1999, and PCTpatent application US99/24483, filed Oct. 20, 1999, each of which areincorporated herein by reference). The plasmids containing the eryAIIImutations in module 5 were pKOS025-1831 (AT?rapAT2), pKOS025-1832(KR→AT/ACP linker), pKOS025-1833 (KR→rapDH/KR4) and pKOS025-1834(KR→rapDH/ER/KR1); and in module 6 were pKOS025-1841(KR→rapDH/KR4),pKOS021-106 (KR→AT/ACP linker) and pKOS025-1842 (AT→rapAT2). These wereconstructed as follows. The BglII-EcoRI fragments containing thecorresponding mutations were transferred into the plasmids described inFIG. 2 from plasmids pKOS006-188, pKOS016-12, pKOS006-178, pKOS026-11b,pKOS011-25, pKOS011-13 and pKOS015-53, respectively (see McDaniel etal., 1999, supra, and U.S. patent application Ser. No. 09/429,349, filedOct. 28, 1999, and PCT patent application US99/24483, filed Oct. 20,1999, each of which are incorporated herein by reference). PlasmidpKOS038-20 contains eryAII with the AT3→rapAT2 replacement in module 3and was made by transferring the mutation from a previously preparedsub-clone pKOS015-28 (see McDaniel et al., 1999, supra, and U.S. patentapplication Ser. No. 09/429,349, filed Oct. 28, 1999, and PCT patentapplication US99/24483, filed Oct. 20, 1999, each of which areincorporated herein by reference).

EXAMPLE 3 Streptomyces Transformation

[0053]Streptomyces lividans K4-114 and K4-155 (see U.S. patentapplication Ser. No. 09/181,833, filed Oct. 28, 1998, and Ziermann etal., 1999, Recombinant polyketide synthesis in Streptomyces: engineeringof improved host strain, BioTechniques 26: 106-110, both of which areincorporated herein by reference) transformants were prepared accordingto standard methods (see Hopwood et al., 1985, supra) using apramycin(100 μg/mL), thiostrepton (50 μg/mL), and hygromycin (225 μg/mL) in theR5 protoplast regeneration plates. In the three-plasmid system for whicheryAII/pSAM2-hyg replaced eryAII/SCP2*-hyg, S. lividans was transformedsequentially with pKOS010-153, pKOS038-67, and pKOS021-30.

EXAMPLE 4 Diketide Feeding

[0054] The (2S, 3R)-2-methyl-3-hydroxyhexanoyl-N-acetylcysteamine (NAC)thioester (propyl diketide) was synthesized in accordance with themethod described in U.S. patent application Serial Nos. 60/117,384,filed Jan. 27, 1999, and Ser. No. 09/492,733 (attorney docket no.30062-20032.00), filed Jan. 27, 2000, by the same inventors and claimingpriority to the foregoing application. The Streptomyces lividans tripletransformants containing the KS1 null allele of eryAI (see Jacobsen etal., 1997, supra, and PCT patent publication Nos. 99/03986 and 97/02358,each of which is incorporated herein by reference) were cultured in 5 mLof R5 medium at 30° C. for 6 days under appropriate antibioticselection. On day 4 of the incubation, 300 μL of diketide solution (4.7mg/mL in 10% DMSO) and 50 μL of pentanoic acid (2.5 mg/mL) were added tothe culture.

EXAMPLE 5 Production and Analysis of Polyketide Analogs

[0055] The Streptomyces lividans triple transformants were cultured in 5mL of R5 medium (see Hopwood et al., 1985, supra) at 30° C. for 6 daysunder appropriate antibiotic selection. The cultured solution wasextracted with 2×5 mL of ethyl acetate and the organic layers werecombined and concentrated. A 50 μL aliquot of the concentrates wasanalyzed by HPLC on a reverse phase C₁₈ column (4.6 mm×15 cm, Beckman,Fullerton, Calif.) using a PE SCIEX API100 LC/MS based detector(Perkin-Elmer, Foster City, Calif.). Quantitative determination ofpolyketide yield was made with evaporative light scattering detection(Analtec model 500 ELSD, Deerfield, Ill.). The polyketides wereidentified by their mass spectrum and correspondence to the productsexpected or to known standards. Under the ionization conditions used,6dEB and its analogs generate signature dehydration patterns. The yieldof the 13-ethyl 6dEB analogs varied from 7 mg/L to less than 0.1 mg/Land the 13-propyl analogs were produced in a range of 0.2 to 20 mg/L.The yields of the 12-membered lactones were not determined.

[0056] The invention having now been described by way of writtendescription and examples, those of skill in the art will recognize thatthe invention can be practiced in a variety of embodiments and that theforegoing description and examples are for purposes of illustration andnot limitation of the following claims.

1. A method for expressing a polyketide or non-ribosomal peptide in ahost cell employing a multiplicity of recombinant vectors, which may beintegrative or freely replicating, wherein each of the multiplicity ofvectors encodes a portion of the polyketide synthase or non-ribosomalpeptide synthase that produces the polyketide or non-ribosomal peptide,said method comprising steps of introducing said vectors into said cell,and culturing said cell into which said vectors have been introducedunder conditions such that said polyketide synthase or non-ribosomalpeptide synthase is produced.
 2. The method of claim 1, wherein saidvectors encode a polyketide synthase.
 3. The method of claim 2, whereinsaid polyketide synthase is selected from the group consisting of DEBS,an oleandolide PKS, a picromycin PKS, an epothilone PKS, a megalomicinPKS.
 4. The method of claim 1, wherein said vectors encode anon-ribosomal peptide synthase.
 5. The method of claim 1, wherein atleast one of the multiplicity of vectors encodes a protein that furthermodifies the polyketide or non-ribosomal peptide produced by saidpolyketide synthase or non-ribosomal peptide synthase.
 6. The method ofclaim 1, wherein at least one of said vectors replicatesextrachromosomally in said host cell.
 7. The method of claim 1, whereinat least one of said vectors integrates into the chromosome of said hostcell.
 8. The method of claim 1, wherein said host cell is a Streptomyceshost cell.
 9. The method of claim 8, wherein said vector is a derivativeof a vector selected from the group consisting of integrating vectorspSET152 and pSAM2 and extrachromosomally replicating vectors SCP2* andpJV1.